The overall goal of this project is to develop xenogenic monoclonal antibody reagents recognizing unique surface antigenic receptors on murine lymphocytes and their precursors. Female Wistar rats were immunized with single cell preparations of whole spleen from New Zealand Black Mice. Hybridoma cell lines were generated by fusing immune rat spleen cells with non secreting NS-1, myeloma cell line. A hybridoma clone VMM-2 was found to secrete an IgG2b antibody recognizing antigenic receptors on cells from bone marrow, spleen but not from thymus by flow microfluorometry. VMM-2 binds specifically to IgM+ B cells. Further, the antibody was found to be specific for the cells of the H-2d haplotype. Genetic mapping using a panel of recombinant inbred and congenic mice, revealed that the antigenic site is located at the K end of the MHC complex. Further studies, using a series of transfected cell lines expressing chimeric H-2Kd gene products on their membranes has confirmed that the antigenic determinant is the region between the residues D152 to S184, located at the C terminal end of the alpha2 domain of the Kd molecule. A comparative Western analyses of lysates from T, B and monocyte cell lines (H-2d) has indicated that in addition to binding to the monomeric form (45000 m.w), of the class I molecules from all the cell types (similar to a conventional anti-Kd), VMM-2 specifically binds to a protein of approximately 180-200,000 m.w. on IgM bearing small B cells (BCL1). Experiments to further confirm the putative antigen recognized by VMM-2 on these cells is being carried out by immune-precipitation of bitotinylated cells and analyses by streptavidin-enzyme conjugate. cDNA clones coding for the putative antigen were isolated by antibody screening of BCL1 cDNA library. A positive clone carrying an insert of approx. 1.6 kb has been identified and being converted into a plasmid for further studies.